Design, synthesis and structure-activity relationship study of novel pyrazole-based heterocycles as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2010.09.054

The versatile hitherto unreported 3-[(E)-3-(dimethylamino)acryloyl]-1,5- diphenyl-1H-pyrazole-4-carbonitrile (3) was prepared via the reaction of 3-acetyl-1,5-diphenyl-1H-pyrazole-4-carbonitrile (1) with dimethylformamid- dimethylacetal (DMF-DMA). The latter product and 3-((E)-3-morpholin-4-yl- acryloyl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile (4) underwent regioselective 1,3-dipolar cycloaddition with nitrilimines to afford the corresponding pyrazole derivatives. In vivo anti-estrogenic activity and acute toxicity after single oral dose of the newly synthesized compounds were evaluated. In vitro disease-oriented primary antitumor screening utilizing 14 cell lines of breast and ovarian tumor subpanels has been also carried out. All tested compounds showed anti-estrogenic properties equipotent or superior to the reference drug, letrozole. 3-[3-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-1-(4-methylphenyl)-1H- pyrazole-4-carbonyl]-1,5-diphenyl-1H-pyrazole-4-carbonitrile (27c) and 3-(3-acetyl-1-phenyl-1H-pyrazole-4-carbonyl)-1,5-diphenyl-1H-pyrazole-4- carbonitrile (8a) showed a significant cytotoxic activity in a nanomolar range against certain types of breast and ovarian tumors with tolerable toxicity.

Full text of DOI:10.1016/j.ejmech.2010.09.054

European Journal of Medicinal Chemistry 45 (2010) 5887e5898
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and structureeactivity relationship study of novel
pyrazole-based heterocycles as potential antitumor agents
,
b
a
c
Ahmad M. Farag a , Korany A.K. Ali , Taha M.A. El-Debss , Abdelrahman S. Mayhoub ,
*
Abdel-Galil E. Amr b 1, Naglaa A. Abdel-Hafez b, Mohamed M. Abdulla d
,
a Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
b Department of Applied Organic Chemistry, National Research Center, Dokki, Giza 12622, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
d Research Units, Hi-Care Pharmaceutical Co., Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The versatile hitherto unreported 3-[(E)-3-(dimethylamino)acryloyl]-1,5-diphenyl-1H-pyrazole-4-car-
bonitrile (3) was prepared via the reaction of 3-acetyl-1,5-diphenyl-1H-pyrazole-4-carbonitrile (1) with
dimethylformamid-dimethylacetal (DMF-DMA). The latter product and 3-((E)-3-morpholin-4-yl-
acryloyl)-1,5-diphenyle1H-pyrazole-4-carbonitrile (4) underwent regioselective 1,3-dipolar cycloaddi-
tion with nitrilimines to afford the corresponding pyrazole derivatives. In vivo anti-estrogenic activity
and acute toxicity after single oral dose of the newly synthesized compounds were evaluated. In vitro
disease-oriented primary antitumor screening utilizing 14 cell lines of breast and ovarian tumor
subpanels has been also carried out. All tested compounds showed anti-estrogenic properties equipotent
or superior to the reference drug, letrozole. 3-[3-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-1-(4-
methylphenyl)-1H-pyrazole-4-carbonyl]-1,5-diphenyl-1H-pyrazole-4-carbonitrile (27c) and 3-(3-acetyl-
1-phenyl-1H-pyrazole-4-carbonyl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile (8a) showed a significant
cytotoxic activity in a nanomolar range against certain types of breast and ovarian tumors with tolerable
toxicity.
Received 11 June 2010
Received in revised form
22 September 2010
Accepted 23 September 2010
Available online 1 October 2010
Keywords:
Anti-estrogenic activity
Antitumor
Cytotoxic activity
Nitrilimines
1,3-Dipolar cycloaddition
1,5-Diphenylpyrazoles
Pyrazolo[3,4-d]pyridazins
Structureeactivity relationship (SAR)
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
It was proved that celecoxib exerts its antitumor action via
inhibition of aromatase, an enzyme complex consisting of a cyto-
Celecoxib (Fig. 1a) is a non-steroidal anti-inflammatory (NSAID)
drug approved for treatment of the signs and symptoms of osteo-
arthritis, rheumatoid arthritis, management of acute pain, treat-
ment of primary dysmenorrhea and to reduce the number of
adenomatous colorectal polyps in Familial Adenomatous Polyposis
(FAP). New indication of celecoxib was also approved for treatment
of signs and symptoms of ankylosing spondylitis [1]. It was cate-
gorized as selective cyclooxygenase-2 (COX-2) inhibitor.
On the other hand, several authors have reported the inhibitory
effects of celecoxib on breast cancer [2e6], recurrent malignant
glioma [7], advanced non-small cell lung cancer [8e10], refractory
multiple myeloma [11], advanced colorectal cancer [12] and
advanced pancreatic cancer [13].
chrome P-450 hemoprotein and a flavoprotein, which converts
C-19 androgens, such as testosterone and androstenedione, to C-18
estrogen such as estradiol and estrone [14e17]. Therefore, it is
useful in controlling the progression of tumors with estrogen
receptors; i.e. breast and ovarian tumors.
In continuation of our recent work aiming at the synthesis of
a variety of heterocyclic ring systems with remarkable biological
importance [18e28] and that related to the structure of celecoxib
and other 1,5-diphenylpyrazole with known antineoplastic effects
[29], we report here our procedure for a regioselective synthesis of
a series of pyrazole-based heterocyclic ring systems with a new
substitution pattern which could reinforce the interaction of 1,5-
diphenylpyrazole skeleton with aromatase enzyme (Fig. 1c).
All the newly synthesized compounds were designed to contain
cyano group at C4 of the pyrazole ring, as it is common in different
aromatase inhibitors such as anastrozole [30,31], fadrozole [32] and
letrozole [33,34] (Fig. 1c). In addition, the bulky polycyclic struc-
tures at C1, 3 and 5 of the pyrazole ring provide favorable
* Corresponding author. Tel.: þ20 12377 3794; fax: þ20 235727556.
E-mail address: afarag49@yahoo.com (A.M. Farag).
1
Present address: Department of Pharmaceutical Chemistry, College of Phar-
macy, King Saud University, Riyadh 11451, Saudi Arabia.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.054

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A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
CF3
O
NC
N
Z
N
N
N
N
N
N
N
N
X
NC
CN
SO2NH2
a
b
c
Fig. 1. Heterocyclic ring systems related to pyrazoles.
interaction with the hydrophobic residues in the largely nonpolar
active sites of the cytochrome isozymes [35].
products were identified as the pyrazole structures 8aec
(Scheme 2). The latter products were assumed to be formed via
initial 1,3-dipolar cycloaddition of the nitrilimines 6aec to the
activated double bond in compound 3 or 4 to afford the non-isolable
cycloadducts 7aec which undergoes loss of dimethylamine or
morpholine molecule yielding the final pyrazole derivatives 8aec.
The other possible regioisomer 9 was excluded on the basis of
the spectral data of the isolated products. For example, in the
pyrazole ring system, C-4 is the most electron-rich carbon, thus H-4
is expected to appear in the NMR spectra at higher field, typically
near 6.0 ppm. On the other hand, H-5 is linked to the carbon
attached to a nitrogen atom and thus it is deshielded to appear near
8.0 ppm. The 1H NMR spectra of the isolated products 8aec
revealed, in each case a singlet signal in the region of 8.54e8.67
which indicates the presence of the pyrazole H-5 rather than H-4.
2. Results and discussion
2.1. Chemistry
In the course of our investigation, we have found that the 3-[(E)-
3-(dimethylamino)acryloyl]-1,5-diphenyl-1H-pyrazole-4-carbon-
itrile (3) is an excellent building block for the synthesis of a variety
of heterocyclic ring systems incorporating a pyrazole moiety. The
enaminone derivative 3 was obtained from the reaction of 3-acetyl-
1,5-diphenyl-1H-pyrazole-4-carbonitrile (1) with dimethylforma-
mid-dimethylacetal (DMF-DMA) (Scheme 1). The structure of
compound 3 was confirmed by its elemental analysis and spectral
data (see Section Experimental).
Treatment of the enaminone 3 with morpholine under reflux
condition affords 3-((E)-3-morpholin-4-yl-acryloyl)-1,5-diphenyle
1H-pyrazole-4-carbonitrile (4) (Scheme 1). The IR spectrum of the
latter product exhibited characteristic bands at 1680 and 2230 cmꢀ1
corresponding to a carbonyl and a nitrile group respectively. Its
mass spectrum revealed a peak at m/z 384 corresponding to its
molecular ion.
O
O
H3C
Cl
Et3N
N
N
N
NH
Ar
Ar
5
6
3
4
or
dry benzene
When either compound 3 or 4 was allowed to react with the
nitrilimines 6, 13, 18, 22 and 26 [liberated in situ from the corre-
sponding hydrazonyl halides 5, 12, 17, 21 and 25, respectively, with
triethylamine in refluxing benzene], it afforded the new pyrazole
derivatives 8aec, 14aec, 19aec, 23aec and 27aec, respectively
(Schemes 2e7).
Thus, nitrilimines 6aec [generated in situ by the action of trie-
thylamine on 2-oxo-N-arylpropane hydrazonyl chlorides 5aec]
reacted with either compound 3 or 4, in refluxing benzene, and
afforded, in each case, only one isolable product. The reaction
O
Y
O
O
O
N
N
NC
NC
N
N
Ar
N
Ar
N
Y
Ph
N
Ph
N
Ph
9
7
Ph
-YH
-YH
O
N
O
O
O
O
O
NC
N
N
N
N
OMe
N
Xylene
reflux
N
NC
N
Ph
+
N
Ar
N
Ar
N
N
Ph
N
Ph
N
N
OMe
Ph
Ph
Ph
N
2
1
3
Ph
10
8
H
N
Ph
Ar
EtOH/
reflux
NH2NH2
O
C6H5
C6H4-pCl
a
b
c
N
N
N
NC
N
O
C6H4-pCH3
N
N
Ph
N
Ph
N
N
O
N
Ph
4
Ar
11
Ph
Y
= NMe2 or
N
O
Scheme 1.
Scheme 2.

A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
5889
O
Ph
Ph
Cl
O
+
Et3N
Cl
Et3N
EtO
N
EtO
N
NH
Ph
21
N
N
N
NH
Ar
N
Ph
Ar
22
13
12
3
4
or
dry benzene
-YH
3 or 4
dry benzene
-YH
O
Ph
Ph
OEt
O
O
O
O
N
N
OEt
NC
O
N
N
N
N
Ar
NC
NC
N
NC
N
N
Ph
Ar
Ph
N
N
N
Ar
N
Ph
N
Ph
N
24
Ph
23
Ph
N
15
Ph
Ph
14
Ph
Y
= NMe2 or
Scheme 5.
N
O
NH2NH2
Ar
H
N
OH
N
O
N
NC
N
NC
a
b
c
C6H5
N
N
Ar
Ph
of the isolated products 14aec showed, in each case, two strong
absorption bands in the region 1720e1660 cmꢀ1 corresponding to
two carbonyl groups. A further confirmation of the proposed
structure came from the reaction of products 14aec with hydrazine
hydrate, in refluxing ethanol, which afforded the corresponding
fused ring systems 16aec. The structures of the products 16aec
were confirmed on the basis of their elemental analysis and spec-
tral data (see Section Experimental).
C6H4-pCl
N
N
Ar
Ph
O
N
N
Ph
N
C6H4-pCH3
N
Ph
16
Y = NMe2 or
N
Scheme 3.
In
a similar manner, phenylcarbamolyl-N-arylnitrilimines
This conclusion was further confirmed chemically by the reaction
compounds 8aec with hydrazine hydrate, to afford fused pyr-
azolopyridazine ring systems 11aec (Scheme 2).
18aec (liberated in situ from phenylcarbomoylhydrazonoyl chlo-
rides 17aec by the action of triethylamine) react with the enami-
none derivative 3 or 4 to furnish the corresponding pyrazole
derivatives 19aec (Scheme 4).
The IR spectra of the isolated products 14aec exhibited, in each
case, two strong absorption bands near 1650 and 1690 cmꢀ1 cor-
responding to two carbonyl groups. The 1H NMR spectrum of
compound 19a, taken as a atypical example of the prepared series,
revealed a signal corresponding to pyrazole H-5 proton at 8.50 ppm
in addition to an aromatic multiplet in the region 7.20e7.37 ppm
Similarly, when of the enaminone 3 or the morpholinyl deriv-
ative 4 was treated with the nitrilimine 13 [generated in situ by the
action of triethylamine on the hydrazonyl chlorides 12aec] they
afforded the corresponding pyrazole derivatives 14aec (Scheme 3).
The structures of the isolated products 14aec were established
on the basis of their elemental analysis and spectral data. For
example, the 1H NMR spectra of compounds 14aec displayed, in
each case, the pyrazole H-5 in the region of 8.61e8.67 ppm which is
in accordance with the proposed structure. Moreover, the IR spectra
O
O
Br
N
+
NC
NC
O
N
N
O
NH
Ar
Ar
N
Ph
Cl
Et3N
Ph
N
Ph
N
H
N
Et3N
N
H
Ph
N
Ph
26
N
N
Ph
NH
Ar
N
25
Ar
3 or 4
dry benzene
-YH
18
17
Ph
CN
N
3
4
or
O
N
N
dry benzene
-YH
Ph
O
O
O
Ph
Ph
O
NC
NC
N
N
O
O
NH
Ph
N
CN
Ph
O
N
H
N
N
Ar
Ph
NC
N
Ph
NC
N
N
N
N
N
N
Ar
Ph
28
Ph
27
N
Ph
N
Ar
N
N
Ar
Ph
N
Ar
NH2NH2
N
Ar
Ph
20
19
Ph
Ph
N
C6H5
C6H4-pCl
C6H4-pCH3
a
C6H5
a
b
c
Ph
N
N
b
c
NC
N
C6H4-pCl
C6H4-pCH3
CN
N
N
Ph
N
Y = NMe2 or
N
O
Ar
Y
Ph
= NMe2 or
N
O
29
Scheme 4.
Scheme 6.

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A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
O
O
Br
N
+
NC
NC
N
N
NH
Ar
Et3N
Ar
N
N
Ph
N
Ph
N
Ph
N
+
Ph
30
Ph
O
31
25
dry benzene
- NHMe2
O
Ph
O
O
Ph
O
N
NC
N
NC
Ph
N
N
Ar
N
Ar
N
Ph
N
N
Ph
33
32
Ph
NH2NH2
Ph
Ar
N
a
b
c
C6H5
C6H4-pCl
C6H4-pCH3
N
NC
N
N
Ph
N
N
Ar
Ph
34
Scheme 7.
and a signal at 10.80 ppm (D2O-exchangeable) characteristic for NH
proton.
Pharmacology and Toxicology Department of National Research
Center, Dokki, Egypt.
Similarly, when the enaminone 3 or 4 was treated with
nitrileimine 22 [generated in situ by the action of triethylamine
on N-phenylbenzohydrazonoyl chloride 21], it afforded 3-(1,3-
diphenyl-1H-pyrazole-4-carbonyl)-1,5-diphenyl-1H-pyrazole-4-
cabonitrile (23) (Scheme 5).
The mass spectrum of the product 23 exhibited a peak corre-
sponding to its molecular ion at m/z 491 and its 1HNMR spectrum
revealed a singlet signal at 8.60 ppm corresponding to pyrazole H-5
proton, in addition to an aromatic multiplet in the region
7.20e7.37 ppm.
Treatmentoftheenaminonederivative3or4withthenitrileimine
derivatives 26aec [generated in situ by the action of triethylamine
onthe pyrazole hydrazonyl bromides25aec] afforded 3-[3-(4-cyano-
1,5-diphenyl-1H-pyrazole-3-yl)-1-aryl-1H-pyrazole-4-caronyl]-1,5-
diphenyl-1H-pyrazole-4-carbonitriles 27aec (Scheme 6).
When the products 27aec were treated with hydrazine hydrate,
they afforded the corresponding 3-[7-(4-cyano-1,5-diphenyl-pyr-
azole-3-yl)-2-aryl-2H-pyrazolo[3,4-d]pyridazin-4-yl]-1,5-diphenyl-
1H-pyrazole-4-carbonitrile derivatives 29aec. The IR spectra of
isolated products 29aec showed, in each case, the disappearance of
bands corresponding to carbonyl groups.
The nitrilimine intermediates 26aec react also with N,N-
dimethylamino-1-phenylpropenone (31) to afford the corresponding
3-(4-benzoyl-1-aryl-1H-pyrazole-3-carbonyl)-1,5-diphenyl-1H-pyr-
azole-4-carbonitriles 32aec. Treatment of the later products with
hydrazine hydrate, afforded the corresponding 34aec (Scheme 7).
In the used method, the increase of uterine weight in castrated
female rats, induced by repeated administration of estradiol, is
antagonized by anti-estrogenic compounds. The test compounds
(>99% pure) in carboxymethylcellulose (CMC) are administered
orally by gavage to groups of immature ovarectomized rat. On the
8th day the animals are sacrificed and the uterine weights were
determined.
The mean value of % reduction in uterine weight was calculated
relative to control group, which was treated with estradiol alone,
and relative potency to reference drug was calculated as depicted in
Table 1.
2.2.2. Acute toxicity (LD50
The rats were dosed by oral gavage with different doses of
aqueous suspensions of very fine powder of the tested
)
a
compounds. Under the conditions of these gavage studies, there
was a great safety for albino rates in comparison with letrozole as
a reference drug as shown in Table 1.
The result showed that, the anti-estrogenic effect of all tested
compounds was found similar or superior to those of reference
drug (Table 1 and Fig. 2). However, the safety varied, some are safer
others are more toxic than letrozole (Table 1 and Fig. 3). The type of
function group at C4 in the pyrazole ring B (Fig. 4) is of great
influence on both anti-estrogenic pharmacological effect and acute
toxicity of the tested compound. Compounds that carry phenyl-
carbamoyl moiety were found 1.6 times to be more potent than the
reference drug (Table 1 and Fig. 3). Removal of NH from such group
has dramatic increase in potency with the p-chloro derivative as
shown in compound 32c (Table 1 and Fig. 2). In contrast, removal of
the carbamoyl moiety decreased the anti-estrogenic activity as
shown in compound 23 (Table 1 and Fig. 2). Also, chlorinated
derivatives are more active than non-halogenated derivatives as
shown in compounds 14c, 19c and 32c (Table 1 and Fig. 2).
2.2. Pharmacology and toxicology
2.2.1. Anti-estrogenic activity
The anti-estrogenic activity of the synthesized compounds and
letrozole (FemaraÒ), as a reference drug, was evaluated in the

A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
5891
Table 1
selectivity against both utilized breast and ovarian cancer cell lines
(Tables 2 and 3). In other words, compounds that revealed good
effect against breast tumor cell lines such as 4, 8a, 11a, 14a, 16a, 19a,
29b and 32b (Table 2) have a moderate to weak activity against
ovarian tumor cell lines (Table 3). In contrast, compounds such as
14c, 16b, 29a, 32c and 34c showed remarkable activity against
ovarian tumors cell lines and a moderate to weak activity against
breast tumor cell lines (Tables 2 and 3). However, compounds 27c
and 34b exhibited an excellent activity against both tumor cell lines
(Tables 2 and 3). With exception of compound 27c, all chloro
derivatives either excluded by NCI or revealed weak activity against
breast tumor cell lines (Table 2).
Mean anti-estrogenic activity and acute toxicity (LD50
compounds and letrozole*.
)
of the synthesized
Compound
% Reduction in
uterine weighta
Relative potency to
LD50 (mg/kg)c
letrozoleb
Letrozole
3
4
21.65
41.98
43.43
32,45
23.97
23.98
34.86
42.83
32.86
23.76
34.87
34.65
23.54
32.43
34.87
43.41
23.87
43,98
32.59
21.87
1
1.93
2
1.49
1.1
1.1
1.61
1.96
1.51
1.09
1.61
1.60
1.08
1.49
1.61
2
252.61 ꢁ 0.11
231.90 ꢁ 0.14
463.11 ꢁ 0.18
323.71 ꢁ 0.11
32.54 ꢁ 0.15
121.73 ꢁ 0.11
142.69 ꢁ 0.12
273.61 ꢁ 0.11
114.18 ꢁ 0.14
322.00 ꢁ 0.10
101.34 ꢁ 0.13
121.91 ꢁ 0.14
121.91 ꢁ 0.14
211.19 ꢁ 0.13
222.00 ꢁ 0.10
111.52 ꢁ 0.12
552.81 ꢁ 0.16
452.81 ꢁ 0.14
13 1.9 ꢁ 0.14
452.81 ꢁ 0.12
8a
11a
14a
14b
14c
16a
16b
19b
19c
23
27c
29a
29b
32b
32c
34a
34c
2.2.3.1. Effect of the newly synthesized compounds on breast can-
cer. Detailed interpretation of the obtained results showed that
compound 8a has activity against MiDA-N tumor cell line in
nanomolar range (GI50 value is 77 nM) (Table 2). The enaminone 4
and the pyrazole ethyl ester 14a are nearly equipotent with
almost the same range of activity against MCF-7 tumor cell line
1.1
2.16
1.5
(GI50 values are 5.49
showed remarkable activity against HS-578T cell line (GI50 values
are 6.4, 1.4 and 8.8 M, respectively) (Table 2). GI50 of compound
16a is less than 3 M against T-47D and NCI/ADR-RES cell lines
mM) (Table 2). Compounds 8a, 11a and 19a
1.01
m
m
*Statistical comparison of the difference between estradiol control group and
treated groups was done by one-way ANOVA and Duncan’s multiple comparison
test *P < 0.05.
(Table 2). Both phenylcarbamoyl 19a and its p-tolyl derivatives
and 19b showed GI50 against MDA/MB-435 cell lines around
2.1 mM (Table 2). Compound 19b revealed good activity against
MiDA-N cell line (Table 2). Removal of NH or carbamoyl moieties
from the latter compound is associated with an increase in
potency against the same cell line as shown in compounds 23 and
a
Value represents mean of twelve rats.
Potency was calculated as regards the percentage change of the letrozole.
Value represents mean values ꢁ SE of five mice per group.
b
c
Compounds that carry ethyl carboxylate, phenylcarbamoyl or
phenyl moieties at C4 of the pyrazole ring B (Fig. 4) are more toxic
than letrozole; while those carrying acetyl or phenylcarbonyl
moieties are safer than the reference drug (Table 1 and Fig. 3).
32b (GI50 values are 5.5, 3.3 and 2.7
mM, respectively) (Table 2). BT
549 tumor cell line was found to be sensitive to compounds 23,
27c and 29b (GI50 values are 1.1, 2.4 and 1.0 mM, respectively)
(Table 2).
Replacement of phenyl ring in compounds 19 with 4-cyano-1,5-
dipehnylpyrazolyl moiety as in case of compound 27c produces the
most active member in this study which revealed antitumor
activity in a nanomolar range against MCF-7 tumor cell line (GI50
value is 61 nM) (Table 2).
2.2.3. In vitro disease-oriented primary antitumor screening
Twenty samples of the newly synthesized compounds were
selected by the National Cancer Institute (NCI), Bethesda, Maryland,
USA, to evaluate their in vitro antitumor activity using 14 breast and
ovarian cell lines. The results revealed high degree of antitumor
2.5
2
1.5
1
0.5
0
Tested Compounds
Fig. 2. Anti-estrogenic potency of the tested compounds relative to letrozole.

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A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
600
550
500
450
400
350
300
250
200
150
100
50
0
Tested Compounds
Fig. 3. LD50 of tested compounds and letrozole after single oral dose.
The benzoyl derivative 32b showed remarkable activity against
six tumor cell lines (GI50 values between 2.6 and 7.1 M) (Table 2),
the latter compound, by replacement of hydroxy group with
phenyl moiety, maintains GI50 values against OVCAR-4 and
m
while its p-chloro isostere 32c has no significant antitumor activity
against the same breast tumor cell lines (Table 2). The pyridazine
derivative 34b maintained the activity against MDA/MB. 231 ATTC,
NCI/ADR-RES and MDA/MB-435 tumor cell lines with little varia-
tions (Table 2). At the same time, it increased the cytotoxic activity
against MCF-7, HS-578T and BT 549 tumor cell lines. Finally, the
cytotoxic activity was decreased against T-47D, MDA/MB-435 and
MiDA-N tumor cell lines (Table 2). The p-chloro derivative 34c
showed weak activity against all tested tumor cell lines (Table 2).
OVCAR-8 cell lines below 10 mM, in addition to an increase in
cytotoxic activity against OVCAR-5 cell line as shown in compound
34b (Table 3).
In addition, compound 23 showed GI50 below 10 mM against
used OVCAR tumor cell lines. The broad spectrum activity was
observed in both compounds 19b and 29a since they possess
remarkable activity against all tested ovarian cell lines (Table 3).
Finally, compound 34c revealed significant activity against all
used tumor cell lines except IGROVI (Table 3).
2.2.3.2. Effect of the newly synthesized compounds on ovarian can-
cer. In general, the effects of the tested compounds against ovarian
tumor cell lines are much less than their effect on breast tumor cell
lines (Table 3). However, compound 27c has an excellent effect in
a nanomolar range against IGROVI ovarian tumor cell line (GI50
value is 22 nM) (Table 3). Enaminones 3, 4 and acetylpyrazole
derivatives 8 have a moderate to weak activity against different
ovarian tumor cell lines (Table 3). The p-chloro derivative 14c
revealed good activity against OVCAR-4 and OVCAR-8 tumor cell
3. Conclusion
The objective of the present study is the synthesis and evalua-
tion of two pharmacological activities, in addition to acute toxicity
study. The first is in vivo investigation of anti-estrogen activity. The
second is in vitro investigation of the antitumor activity on breast
and ovarian cell lines of new 1,5-diphenylpyrazoles incorporating
cyano pharmacophore group, structurally related to well-docu-
mented antineoplastic agents. This aim has been verified by the
synthesis of hybrid compounds comprising the above-mentioned
pharmacophores substituted essentially with
ylphenylpyrazole counterpart at the pyrazole-C3 moiety having the
general structures shown in Fig. 1c.
The highest active derivative in present study is compound 27c
which showed significant cytotoxic activity in a nanomolar range
against certain types of breast and ovarian tumors with tolerable
toxicity (LD50 > 200 mg/kg). In addition, its anti-aromatase activity
was found one and half times more than letrozole.
lines (GI50 values are 5.4 and 9.1 mM, respectively) (Table 3).
Replacement of ethoxy group with phenyl moiety maintains the
antitumor activity against OVCAR-4 tumor cell line (GI50 value id
5.4) as shown in compound 32c (Table 3).
The hydroxypyridazine derivative 16b showed good activity
against OVCAR-4 and OVCAR-8 tumor cell lines (GI50 value are 3.2
a
carbon-
and 4.3 mM, respectively) (Table 3). The increase of lipophilicity of
O
NC
Z
The oral LD50 values in this study revealed that most of the
tested compounds are relatively nontoxic, the LD50 of 11
compounds were found more than 200 mg/kg.
A
N
B
N
N
N
Finally, the remarkable antitumor activity associated with
significant anti-aromatase activity and relatively high safety
margin displayed by these compounds will be of interest for future
structure optimization with the hope of finding more active and
selective antitumor agents.
X
Fig. 4. Compounds carrying ethyl carboxylate, phenylcarbamoyl or phenyl moieties at
C4 of the pyrazole ring.

A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
Growth inhibitory concentration (GI50, mM) of tested compounds on different Breast Cancer’s cell lines.
5893
Table 2
Compound
Cell lines
MDA, MB.
231, ATTC
MCF-7
T-47D
NCI, ADR-RES
HS-578T
MDA, MB-435
MiDA-N
BT 549
3
4
8a
50.4
I.A.a
I.A.a
I.A.a
N.T.b
I.A.a
I.A.a
6.21
I.A.a
I.A.a
6.27
49.8
I.A.a
52.4
7.90
6.12
I.A.a
8.84
6.42
52.1
0.15
39.8
5.49
25.7
8.46
5.49
I.A.a
I.A.a
I.A.a
I.A.a
I.A.a
34.4
33.3
0.061
17.8
9.42
I.A.a
I.A.a
9.53
43.8
41.6
0.7
30.2
I.A.a
36.8
9.14
I.A.a
I.A.a
I.A.a
2.95
I.A.a
33.0
30.8
28.0
25.7
81.0
16.0
7.18
I.A.a
21.8
38.0
42.6
0.44
90.6
I.A.a
I.A.a
I.A.a
I.A.a
I.A.a
I.A.a
2.76
I.A.a
I.A.a
2.48
98.0
I.A.a
24.5
7.46
2.58
I.A.a
I.A.a
2.28
74.7
1.16
I.A.a
I.A.a
6.46
1.39
N.T.b
65.0
I.A.a
46.8
I.A.a
8.84
63.2
I.A.a
37.4
94.4
9.72
I.A.a
I.A.a
31.8
16.8
38.6
2.17
I.A.a
I.A.a
I.A.a
I.A.a
I.A.a
71.8
I.A.a
21.8
I.A.a
2.03
2.23
61.0
I.A.a
I.A.a
9.20
2.65
I.A.a
31.8
23.8
46.3
0.067
45.4
38.4
0.077
38.4
38.4
62.3
I.A.a
13.8
I.A.a
I.A.a
5.49
3.36
26.8
I.A.a
2.26
2.74
I.A.a
31.2
63.8
I.A.a
1.61
31.1
41.4
I.A.a
96.8
31.8
39.8
I.A.a
44.8
I.A.a
11.8
I.A.a
1.12
2.47
11.8
1.02
I.A.a
I.A.a
21.8
38.8
I.A.a
0.89
11a
14a
14b
14c
16a
16b
19a
19b
23
27c
29a
29b
32b
32c
34a
34b
34c
Letrozole
a
I.A., inactive; GI50 value > 100 mM.
N.T., not tested.
b
4. Experimental
4.1. Chemistry
4.1.1. General
All melting points were measured on a Gallenkamp melting
point apparatus (Weiss-Gallenkamp, London, UK). The infrared
spectra were recorded in potassium bromide disks on a pye Unicam
SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometers
(Pye Unicam Ltd. Cambridge, England and Shimadzu, Tokyo, Japan,
respectively). The NMR spectra were recorded on a Varian Mercury
VX-300 NMR spectrometer (Varian, Palo Alto, CA, USA). 1H spectra
were run at 300 MHz and 13C spectra were run at 75.46 MHz in
deuterated chloroform (CDCl3) or dimethyl sulphoxide (DMSO-d6).
Chemical shifts are given in parts per million and were related to
that of the solvent. Mass spectra were recorded on a Shimadzu
GCMS-QP 1000 EX mass spectrometer (Shimadzu) at 70 eV.
Elemental analyses were carried out at the Micro-analytical Center
of Cairo University, Giza, Egypt.
2-Oxo-N-arylpropanehydrazonoyl chlorides 5aec [36], ethyl
2-(2-arylhydrazono)-2-chloroaceatate derivatives 12aec [37] phe-
nylcarbamoylarylhydrazonoyl chlorides 17aec [38], N-phenyl-
benzenecarbohydrazonyl chloride (21) [39], 3-acetyl-1,5-diphenyl-
1H-pyrazole-4-carbonitrile (1) and 4-cyano-a-oxo-1,5-diphenyl-N-
aryl-1H-pyrazole-3-ethanehydrazonoyl bromide derivatives 25aec
[40], and 3-dimethylamino-1-phenylpropenone (31) [41] were
prepared following the procedures reported in the literature.
Table 3
Growth inhibitory concentration (GI50, mM) of tested compounds on different
ovarian cancer’s cell lines.
Compound Cell lines
4.1.2. 3-[(E)-3-(Dimethylamino)acryloyl]-1,5-diphenyl-1H-
pyrazole-4-carbonitrile (3)
IGROVI OVCAR-3 OVCAR-5 OVCAR-8 OVCAR-4 SK-OV-3
3
4
8a
98.0
88.0
96.8
92.0
I.A.a
93.2
94.8
92.2
91.34
99.5
16.6
I.A.a
98.8
21.5
66.8
96.8
N.T.b
I.A.a
91.8
I.A.a
33.4
23.8
7.66
4.94
99.8
7.26
95.8
38.4
63.8
93.8
I.A.a
I.A.a
5.87
I.A.a
90.8
34.8
I.A.a
N.T.b
61.2
I.A.a
61.8
32.8
I.A.a
9.72
6.23
36.8
9.12
I.A.a
36.8
36.8
I.A.a
8.94
7.94
1.62
75.8
I.A.a
62.8
I.A.a
I.A.a
65.8
9.18
61.5
3.23
I.A.a
9.52
9.83
20.3
9.82
61.8
I.A.a
I.A.a
68.0
9.23
9.23
4.50
63.8
62.8
69.8
56.4
I.A.a
45.8
5.46
26.38
4.38
28.4
7.56
5.49
30.4
7.46
43.8
63.8
5.49
63.0
5.49
7.66
0.88
23.8
33.8
39.8
36.8
N.T.b
30.8
I.A.a
36.8
30.8
16.8
9.42
I.A.a
38.8
5.72
36.8
36.8
I.A.a
32.8
I.A.a
9.72
1.09
A mixture of the acetylpyrazole 1 (5.74 g, 20 mmol) and dime-
thylformamide dimethylacetal (DMF-DMA) (2.66 mL, 20 mmol) in
dry xylene (30 mL) was refluxed for 3 h, then allowed to cool. The
orange yellow precipitate was filtered off, washed with petroleum
ether (60/80 ꢂC), dried and crystallized from ethanol/DMF to afford
11a
14a
14b
14c
16a
16b
19a
19b
23
27c
29a
29b
32b
32c
34a
34b
34c
Letrozole
compound 3. Yield 92%; mp 225e227 ꢂC. IR (KBr)
n
cm1: 2230
(C^N), 1693 (C]O). 1H NMR (DMSO-d6)
d
2.55 (s, 6H, 2CH3), 5.85
(d, 1H, CH, J ¼ 12.5 Hz), 7.39 (m, 10H, ArH’s), 7.68 (d, 1H, CH,
J ¼ 12.5 Hz). MS m/z (%): 342 (Mþ, 22.5), 272 (16.3), 180 (9.0), 123
(55.4), 77 (100). Anal. Calcd for C21H18N4O (342.40): C, 73.67; H,
5.30; N, 16.36%. Found: C, 73.73; H, 5.33; N, 16.35%.
0.022
13.3
98.0
I.A.a
4.1.3. 3-((E)-3-Morpholin-4-yl-acryloyl)-1,5-diphenyle1H-
pyrazole-4-carbonitrile (4)
I.A.a
94.8
93.4
93.4
0.095
A solution of the enaminone 3 (3.42 g, 10 mmol) and morpho-
line (0.84, 10 mmol) in ethanol (15 mL) was heated under reflux
condition for 2 h. The reaction mixture was concentrated in
vacuum. The solid product obtained upon cooling was filtered off
and crystallized from ethanol/DMF to give compound 3-((E)-3-
a
I.A., inactive; GI50 value > 100 mM.
N.T., not tested.
b

5894
A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
morpholin-4-yl-acryloyl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile
(4). Yield 70%; mp 172 ꢂC. IR (KBr) v cmꢀ1: 2230 (C^N), 1680 (C]
7.20e7.37 (m, 15H, ArH’s), 8.67 (s,1H, pyrazole-H-5); 13C NMR (DMSO-
d6) 18.04 (CH3), 113.69, 115.58 (pyrazole carbons), 117.95 (C^N),
120.46, 120.53, 121.77, 125.13, 125.57, 128.80, 128.97, 129.15, 129.34,
129.69, 130.29, 138.07 (aromatic carbons), 152.91, 153.98 (pyridazine
C]N carbons). MS m/z (%): 453 (Mþ, 100), 284 (57.1), 77 (72.4). Anal.
Calcd for C28H19N7 (453.50): C, 74.16; H, 4.22; N, 21.62%. Found: C,
74.20; H, 4.23; N, 21.59%.
d
O). 1H NMR (DMSO-d6)
d
2.87 (t, 4H, 2CH2), 3.98 (t, 4H, CH2), 5.81 (d,
1H, CH, J ¼ 12.6 Hz), 7.41e7.69 (m, 10H, ArH’s), 7.89 (d, 1H, CH,
J ¼ 12.6 Hz). MS m/z (%): 384 (Mþ, 72.5), 367 (100), 337 (52.8), 297
(16.6), 272 (50.5),180 (32.1),141 (18.1),112 (35.1), 77 (57.1). Analysis
for: C23H20N4O2 (384.44): C, 71.86; H, 5.24; N, 14.57; S,%. Found: C,
71.90; H, 5.30; N, 14.53%.
4.1.5.3. 3-[7-Methyl-2-(4-chlorophenyl)-2H-pyrazolo[3,4-d]pyridazin-
4.1.4. 3-(3-Acetyl-1-aryl-1H-pyrazole-4-carbonyl)-1,5-diphenyl-
1H-pyrazole-4-carbonitriles 8aec
4-yl]-1,5-diphenyl-1H-pyrazole-4-carbonitrile (11b). Yield 80%; mp:
>300 ꢂC. IR (KBr) v cmꢀ1: 2233 (CN). 1H NMR (DMSO-d6)
d 2.17 (s,
4.1.4.1. General procedure. To a mixture of the enaminone deriva-
tive 3 or 4 (10 mmol) and the appropriate 2-oxo-N-arylpropane-
3H, CH3), 7.30e7.41 (m,14H, ArH’s), 8.85 (s,1H, pyrazole-H-5). MS m/
z: 489 (Mþ, 31.9), 487 (Mþ, 100), 284 (43.4), 244 (32.1), 77 (56.4).
Anal. Calcd for C28H18N7Cl (487.95): C, 68.92; H, 3.72; N, 20.09%.
Found: C, 68.95; H, 3.75; N, 20.11%.
hydrazonoyl chloride
5 (10 mmol) in benzene (20 mL), an
equivalent amount of triethylamine was added. The reaction
mixture was heated under reflux for 8 h. The solvent was distilled
off at reduced pressure and the residual brown viscous liquid was
taken in methanol and the resulting solid was collected by filtration
washed thoroughly with ethanol, dried and finally crystallized from
ethanol to afford corresponding 3-(3-acetyl-1-aryl-1H-pyrazole-4-
carbonyl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile derivative 8.
The synthesized compounds 8aec together with their physical and
spectral data are listed below.
4.1.5.4. 3-[7-Methyl-2-(4-methylphenyl)-2H-pyrazolo[3,4-d]pyridazin-
4-yl]-1,5-diphenyl-1H-pyrazole-4-carbonitrile (11c). Yield 70%; mp:
261e262 ꢂC. IR (KBr) v cmꢀ1: 2229 (C^N). 1HNMR (DMSO-d6)
d 2.14
(s, 3H, CH3), 2.35 (s, 3H, CH3), 7.19e7.38 (m, 14H, ArH’s), 8.71 (s,1H, py
H-5). MS m/z: 467 (Mþ, 100), 284 (50.0), 244 (21.7), 77 (80.6). Anal.
Calcd for C29H21N7 (467.53): C, 74.50; H, 4.52; N, 20.97%. Found: C,
74.51; H, 4.55; N, 20.8%.
4.1.4.2. 3-(3-Acetyl-1-phenyl-1H-pyrazole-4-carbonyl)-1,5-diphenyl-
4.1.6. 3-(1-Aryl-3-ethoxycarbonyl-4-carbonyl)-1,5-diphenyl-1H-
pyrazole-4-carbonitriles 14aec
1H-pyrazole-4-carbonitrile (8a). Yield 74%; mp 160 ꢂC. IR (KBr) v
cmꢀ1: 2223 (C^N), 1708, 1670 (2C]O). 1H NMR (DMSO-d6)
d
2.10
(s, 3H, CH3), 8.67 (s,1H, py H-5), 7.39e7.49 (m,15H, ArH’s); 13C NMR
(DMSO-d6) 27.67 (COCH3), 112.90, 113.85 (pyrazole carbons),
4.1.6.1. General procedure. To a mixture of the appropriate enami-
none derivative 3 or 4 (10 mmol) and the corresponding chloro
(arylhydrazono)ethyl acetate 12 (10 mmol) in benzene (20 mL), an
equivalent amount of triethylamine was added, and the reaction
mixture was heated under reflux for 5 h. The solvent was distilled
off under reduced pressure and the residual viscous liquid was
taken in methanol. The resulting solids were collected by filtration,
washed with ethanol, dried and finally crystallized from ethanol to
afford the compounds 14aec. The physical and spectral data for
obtained product are listed below.
d
119.67 (C^N), 121.00, 125.58, 126.30, 128.18, 128.77, 129.18, 129.30,
133.09, 138.35, 149.68, 150.22, 150.62 (aromatic and pyrazole
carbons), 180.06, 193.43 (C]O carbons). MS m/z (%): 457 (Mþ, 5.4),
442 (54.4), 213 (20.8), 77 (100). Anal. Calcd for C28H19N5O2
(457.50): C, 73.52; H, 4.19; N, 15.31%. Found: C, 73.59; H, 4.23; N,
15.33%.
4.1.4.3. 3-[3-Acetyl-1-(4-chlorophenyl)-1H-pyrazole-4-carbonyl]-1,5-
diphenyl-1H-pyrazole-4-carbonitrile (8b). Yield 70%; mp 221e222 ꢂC.
IR (KBr) v cmꢀ1: 2230 (C^N), 1700, 1663 (2C]O). MS m/z (%): 493
(Mþ, 9.8), 491 (Mþ, 30.2), 476 (46.5), 272 (24.3), 247 (25.3), 205
(88.9), 180 (26.9), 141 (36.5), 77 (100). Anal. Calcd for C28H18N5O2Cl
(491.93): C, 68.36; H, 3.68; N, 14.23%. Found: C, 68.41; H, 3.70; N,
14.26%.
4.1.6.2. 3-(1-Phenyl-3-ethoxycarbonyl-4-carbonyl)-1,5-diphenyl-1H-
pyrazole-4-carbonitrile (14a). Yield 73%; mp 205e206 ꢂC. IR (KBr) v
cmꢀ1: 2239 (C^N), 1732, 1650 (2C]O). 1H NMR (CDCl3)
d 1.23 (t,
3H, CH3, J ¼ 7.2 Hz), 4.29 (q, 2H, CH2, J ¼ 7.2 Hz), 7.20e7.64 (m, 15H,
ArH’s), 8.67 (s, 1H, pyrazole H-5). MS m/z (%): 487 (Mþ, 47), 443
(68.6), 272 (27.5), 215 (41.9), 180 (22.0), 77 (100). Anal. Calcd for
C29H21N5O3 (487.51): C, 71.44; H, 4.34; N,14.36%. Found: C, 71.41; H,
4.33; N, 14.39%.
4.1.4.4. 3-[3-Acetyl-1-(4-methylphenyl)-1H-pyrazole-4-carbonyl]-1,5-
diphenyl-1H-pyrazole-4-carbonitrile(8c). Yield 80%; mp 230e231 ꢂC.
IR (KBr) v cmꢀ1: 2231 (C^N),1700,1662 (2C]O) 1HNMR (DMSO-d6)
4.1.6.3. 3-(1-(4-Chlorophenyl)-3-ethoxycarbonyl-4-carbonyl)-1,5-
d
2.42 (s, 3H, CH3), 2.58 (s, 3H, CH3), 7.22e7.63 (m, 14H, ArH’s), 8.54
diphenyl-1H-pyrazole-4-carbonitrile
215e216 ꢂC. IR (KBr) v cmꢀ1: 2230 (C^N), 1738, 1660 (2C]O). 1H
NMR (DMSO-d6)
J ¼ 7.2 Hz), 7.25e7.61 (m, 14H, ArH’s), 8.62 (s, 1H, pyrazole H-5).
MS m/z (%): 523 (7.2), 521 (Mþ, 23.1), 249 (21), 180 (19), 138 (45),
77 (100). Anal. Calcd for C29H20N5O3Cl (521.97): C, 66.73; H, 3.86;
N, 13.41%. Found: C, 66.75; H, 3.88; N, 13.40%.
(14b). Yield 70%; mp
(s, 1H, py H-5). MS m/z (%): 471 (Mþ, 19.1), 456 (34.2), 227 (15.7), 77
(100). Anal. Calcd for C29H21N5O2 (471.52): C, 73.87; H, 4.48; N,
14.85%. Found: C, 73.90; H, 4.50; N, 14.82%.
d
1.26 (t, 3H, CH3, J ¼ 7.2 Hz), 4.25 (q, 2H, CH2,
4.1.5. Reaction of 3-(3-acetyl-1-aryl-1H-pyrazole-4-carbonyl)-1,5-
diphenyl-1H-pyrazole-4-carbonitriles 8aec with hydrazine hydrate
4.1.5.1. General procedure. Hydrazine hydrate (80%, 2 mL) was
added to a solution of the appropriate compound 8 (5 mmol) in
ethanol (10 mL). The reaction mixture was heated under reflux for
1 h, concentrated in vacuum, and diluted with water. The precipi-
tate obtained was filtered off, washed with ice-cold water, dried
and crystallized from ethanol. The synthesized compounds 11aec
together with their physical and spectral data are listed below.
4.1.6.4. 3-[1-(4-Methylphenyl)-3-ethoxycarbonyl-4-carbonyl]-1,5-
diphenyl-1H-pyrazole-4-carbonitrile
185e186 ꢂC. IR (KBr) v cmꢀ1: 2231(C^N), 1730, 1665 (2C]O). 1H
NMR (DMSO-d6)
1.23 (t, 3H, CH3, J ¼ 7.2 Hz), 2.40 (s, 3H, CH 3),
4.29 (q, 2H, CH2, J ¼ 7.2 Hz), 7.23e7.64 (m, 14H, ArH’s), 8.61 (s, 1H,
pyrazole-H-5); 13C NMR (DMSO-d6)
13.66 (CH3CH2), 20.47 (Ar-
(14c). Yield
72%;
mp
d
d
4-CH3), 61.26 (CH3CH2), 112.76, 113.84 (pyrazole carbons), 119.64
(C^N), 120.56, 125.56, 126.31, 128.77, 129.05, 129.18, 129.26,
129.379, 129.57, 132.69, 136.08, 137.70, 138.16, 144.90, 150.28,
150.70 (pyrazole and aromatic carbons), 161.59 (ester C]O),
4.1.5.2. 3-(7-Methyl-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-4-yl)-1,5-
diphenyl-1H-pyrazole-4-carbonitrile (11a). Yield 70%; mp 280e282 ꢂC.
IR (KBr) v cmꢀ1: 2226 (C^N). 1H NMR (DMSO-d6)
d 2.15 (s, 3H, CH3),

A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
5895
179.13 (ketone C]O).. MS m/z (%): 501 (Mþ, 12.3), 456 (71.1), 428
(23.5), 272 (20.1), 77 (100). Anal. Calcd for C30H23N5O3 (501.55):
C, 71.84; H, 3.72; N, 13.96%. Found: C, 71.89; H, 3.70; N, 14.00%.
Anal. Calcd for C33H22N6O2 (534.57): C, 74.14; H, 4.14; N, 15.72%.
Found: C, 74.18; H, 4.11; N, 15.75%.
4.1.8.3. 4-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-carbonyl)-1-(4-
chlorophenyl)-1H-pyrazole-3-carboxylic acid phenylamid (19b).
Yield 80%; mp 224e225 ꢂC. IR (KBr) v cmꢀ1: 3265 (NH), 2233
4.1.7. Reaction of 3-(1-aryl-3-ethoxycarbonyl-4-carbonyl)-1,5-
diphenyl-1H-pyrazole-4-carbonitriles 14aec with hydrazine
hydrate
(C^N), 1685, 1650 (2C]O). 1HNMR (DMSO-d6)
d: 7.25e7.90 (m,
4.1.7.1. General procedure. Hydrazine hydrate (80%, 3 mL) was
added to a solution of the appropriate 3-(1-aryl-3-ethoxycarbonyl-
4-carbonyl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile 14 (5 mmol)
in ethanol (10 mL). The reaction mixture was heated under reflux
for 1 h, concentrated in vacuum, cooled, and diluted with water.
The precipitate obtained was filtered, washed with ice-cold water,
dried and crystallized from ethanol. The synthesized compounds
16aec together with their physical and spectral data are listed
below.
19H, ArH’s), 9.25 (s, 1H, pyrazole-H-5), 11.47 (br s, 1H, NH, D2O-
exchangeable). MS m/z (%): 570 (Mþ, 6.8), 568 (Mþ, 19.1), 476
(88.1), 272 (22.1) 180 (8.5), 141 (25), 77 (100). Anal. Calcd for
C33H21N6O2Cl (569.02): C, 69.65; H, 3.72; N, 14.77%. Found: C,
69.69; H, 3.70; N, 14.76%.
4.1.8.4. 4-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-carbonyl)-1-(4-
methlphenyl)-1H-pyrazole-3-carboxylic acid phenyl amid (19c).
Yield 72%; mp 240e242 ꢂC. IR (KBr) v cmꢀ1: 3260 (NH), 2230
(C^N), 1683, 1645 (2C]O). 1HNMR (DMSO-d6)
d 2.40 (s, 3H,
4.1.7.2. 3-(7-Hydroxy-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-4-yl)-
1,5-diphenyl-1H-pyrazole-4-carbonitrile (16a). Yield 70%; mp
249e250 ꢂC. IR (KBr) v cmꢀ1: 3350 (OH), 3205 (NH), 2229 (C^N),
CH3), 7.15e7.93 (m, 19H, ArH’s), 9.20 (s, 1H, pyrazole-H-5), 11.55
(br s, 1H, NH, D2O-exchangeable). MS m/z (%): 548 (Mþ, 5.5), 456
(100), 428 (17.6), 244 (43.9), 77 (87.2). Anal. Calcd for C34H24N6O2
(548.61): C, 74.44; H, 4.41; N, 15.32%. Found: C, 74.47; H, 4.45; N,
15.36%.
1678 (C]O). 1H NMR (DMSO-d6)
d 2.98 (br s, 1H, OH, D2O-
exchangeable), 7.17e7.39 (m, 15H, ArH’s), 8.64 (s, 1H, pyrazole H-5),
11.00 (br s, 1H, NH, D2O-exchangeable). MS m/z (%): 455 (Mþ, 100),
398 (33.7), 295 (14.1), 180 (11.1), 77 (95.9). Anal. Calcd for
C27H17N7O (455.92): C, 73.83; H, 3.76; N, 21.52%. Found: C, 73.85; H,
3.77; N, 21.55%.
4.1.9. 3-(1,3-Diphenyl-1H-pyrazole-4-carbonyl)-1,5-phenyl-1H-
pyrazole-4-carbonitrile (23)
To a mixture of the appropriate enaminone derivative 3 or 4
(10 mmol) and N-phenylbenzenecarbohydrazonoyl chloride (21)
(10 mmol) in benzene (30 mL), an equivalent amount of triethyl-
amine was added and the reaction mixture was heated under reflux
for 8 h. The solvent was removed under reduced pressure and the
residual viscous liquid was taken in methanol. The resulting solid
was collected by filtration washed with ethanol, dried and finally
crystallized from ethanol to afford the compound 23. Yield 55%; mp
225e226 ꢂC. IR (KBr) v cm1: 2228 (C^N), 1671 (C]O). 1H NMR
4.1.7.3. 3-[7-Hydroxy-2-(4-chlorophenyl)-2H-pyrazolo[3,4-d]pyr-
idazin-4-yl]-1,5-diphenyl-1H-pyrazole-4-carbonitrile (16b). Yield
72%; mp 288e290 ꢂC. IR (KBr) v cmꢀ1: 3371 (OH), 3230 (NH),
2228 (C^N), 1680 (C]O). 1HNMR (DMSO-d6)
d 3.18 (br s, 1H, OH,
D2O-exchangeable), 7.29e7.50 (m, 14H, ArH’s), 8.71 (s, 1H, pyr-
azole H-5), 11.01 (br s, 1H, NH, D2O-exchangeable). MS m/z (%):
491 (Mþ, 30.8), 489 (Mþ, 100), 434 (10.7), 432 (29.9), 77 (44.2).
Anal. Calcd for C27H16N7OCl (489.92): C, 66.19; H, 3.29; N,
20.01%. Found: C, 66.21; H, 3.32; N, 20.03%.
(DMSO-d6)
d
7.20e7.37 (m, 20H, ArH’s), 8.60 (s, 1H, pyrazole H-5);
113.01, 118.77 (C^N), 120.61, 125.80, 126.29,
13C NMR (DMSO-d6)
d
127.64, 128.66, 129.18, 129.28, 129.71, 130.55, 135.28, 138.60, 143.66,
151.2 (pyrazole and aromatic carbons),178.85 (ketone C]O). MS m/
z (%): 491 (Mþ, 29.1), 247 (26.2), 116 (10.8), 77 (100). Anal. Calcd for
C32H21N5O (491.56): C, 78.19; H, 4.30; N, 14.24%. Found: C, 78.14; H,
4.33; N, 14.26%.
4.1.7.4. 3-[7-Hydroxy-2-(4-methylphenyl)-2H-pyrazolo[3,4-d]pyridazin-
4-yl]-1,5-diphenyl-1H-pyrazole-4-carbonitrile (16c). Yield 75%; mp
295e297 ꢂC. IR (KBr) v cmꢀ1: 3354 (OH), 3200 (NH), 2225 (C^N), 1672
(C]O). 1HNMR (DMSO-d6)
d 2.5 (s, 3H, CH3), 3.10 (br s, 1H, OH, D2O-
exchangeable), 7.20e7.37 (m, 14H, ArH’s), 8.60 (s, 1H, pyrazole-H-5),
10.51 (br s, 1H, NH, D2O-exchangeable). MS m/z (%): 469 (100), 412
(33.5), 244 (8.2), 77 (60.0). Anal. Calcd for C28H19N7O (469.51): C, 71.63;
H, 4.07; N, 20.88%. Found: C, 71.68; H, 4.08; N, 20.86%.
4.1.10. 3-[3-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-1-aryl-1H-
pyrazole-4-carbonyl]-1,5-diphenyl-1H-pyrazole-4-carbonitriles
27aec
4.1.10.1. General procedure. To a mixture of the appropriate enami-
none derivative 3 or 4 (10 mmol) and the corresponding pyr-
azolehydazonyl bromide derivative 25(10 mmol) inbenzene (30 mL),
an equivalent amount of triethylamine was added. The reaction
mixture was heated under reflux for 9 h. The solvent was removed
under reduced pressure. The residual brown viscous liquid was taken
in methanol then the resulting solid was collected by filtration
washed with ethanol, dried and finallycrystallized from ethanol/DMF
to afford the corresponding 3-[3-(4-cyano-1,5-diphenyl-1H-pyr-
azole-3-yl)-1-aryl-1H-pyrazole-4-carbonyl]-1,5-diphenyl-1H-pyr-
azole-4-carbonitrile derivatives 27. The physical and spectral data for
obtained products are listed below.
4.1.8. 4-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-carbonyl)-1-aryl-
1H-pyrazole-3-carboxylic acid phenylamids 19aec
4.1.8.1. General procedure. To a mixture of the appropriate enami-
none derivative 3 or 4 (10 mmol) and the corresponding phenyl-
carbamoylarylhydrazonoyl chloride 17 (10 mmol) in benzene
(30 mL), an equivalent amount of triethylamine was added and the
reaction mixture was heated under reflux for 6 h. The solvent was
removed under reduced pressure and the residual viscous liquid
was taken in methanol. The resulting solid was collected by filtra-
tion washed with ethanol, dried and finally recrystallized from
ethanol to afford the compounds 19aec. The physical and spectral
data for obtained product are listed below.
4.1.10.2. 3-[3-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-1-phenyl-1H-
pyrazole-4-carbonyl]-1,5-diphenyl-1H-pyrazole-4-carbonitrile (27a).
Yield 73%; mp 279e280 ꢂC. IR (KBr) v cmꢀ1: 2223, 2231 (2CN), 1678,
4.1.8.2. 4-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-carbonyl)-1-
phenyl-1H-pyrazole-3-carboxylic acid phenylamid (19a). Yield 73%;
mp 160e161 ꢂC. IR (KBr) v cmꢀ1: 3261 (NH), 2223 (C^N), 1690,
1663 (2C]O).1H NMR (DMSO-d6)
d
7.18e7.40 (m, 25H, ArH’s), 7.93 (s,
1650 (2C]O). 1H NMR (DMSO-d6)
d
7.20e7.37 (m, 20H, ArH’s), 8.50
1H, pyrazole H-5); 13C NMR (DMSO-d6)
d
112.42, 112.53, 119.94
(s, 1H, pyrazole H-5), 10.80 (br s, 1H, NH, D2O-exchangeable). MS m/
(C^N), 123.27, 125.12, 125.44, 128.37, 128.90, 129.20, 129.59, 132.63,
138.29, 149.89, 150.29, 150.349 (pyrazole and aromatic carbons),
z (%): 534 (Mþ, 19.1), 442 (100), 272 (22.4), 141 (13.3), 77 (50.6).

5896
A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
179.013, 180.46 (C]O carbons). MS m/z (%): 686 (Mþ, 4.2) 442 (40.3),
414 (13.2), 272 (50.1), 244 (18.2), 77 (100). Anal. Calcd for C43H26N8O2
(686.73): C, 75.30; H, 3.81; N, 16.31%. Found: C, 75.33; H, 3.79; N,
16.30%.
4.1.11.4. 3-[7-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-2-(4-meth-
ylphenyl)-2H-pyrazolo[3,4-d]pyridazin-4-yl]-1,5-diphenyl-1H-pyr-
azole-4-carbonitrile (29c). Yield 70%; mp >300 ꢂC; IR (KBr) v
cmꢀ1: 2238, 2215 (2 CN). 1H NMR (DMSO-d6)
d 2.43 (s, 3H, CH3),
7.25e7.47 (m, 24H, ArH’s), 9.15 (s, 1H, py H-5). MS m/z (%): 696
(Mþ, 12.6), 244 (3.5), 77 (100). Anal. Calcd for C44H28N10 (696.77):
C, 75.84; H, 4.05; N, 20.10%. Found: C, 75.83; H, 4.08; N, 20.12%.
4.1.10.3. 3-[3-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-1-(4-chlor-
ophenyl)-1H-pyrazole-4-carbonyl]-1,5-diphenyl-1H-pyrazole-4- ꢀ1
2230, 2220 (2CN), 1680, 1631 (2C]O). 1H NMR (DMSO-d6)
carbonitrile (27b). Yield 80%; mp 250e252 ꢂC. IR (KBr) v cm
:
4.1.12. 3-(4-Benzoyl-1-aryl-1H-pyrazole-3-carbonyl)-1,5-diphenyl-
1H-pyrazole-4-carbonitriles 32aec
d
7.25e7.48 (m, 24H, ArH’s), 7.94 (s, 1H, pyrazole H-5). MS m/z (%):
722 (Mþ, 10.8), 720 (Mþ, 33.4), 478 (8.5), 476 (19.8), 272 (72.9), 180
(24.6), 141 (54.1), 77 (100). Anal. Calcd for C43H25N8O2Cl (720.17):
C, 71.61; H, 3.49; N, 15.53%. Found: C, 71.63; H, 3.48; N, 15.55%.
4.1.12.1. General procedure. To a mixture of the enaminone 31
(10 mmol) and appropriate hydrazonyl bromide 25 (10 mmol) in
benzene (30 mL), an equivalent amount of triethylamine was
added. The reaction mixture was heated under reflux for 9 h. The
solvent was distilled off under reduced pressure. The residual
viscous liquid was taken in methanol and the resulting solid was
collected by filtration washed with ethanol, dried and finally
crystallized from ethanol/DMF to afford the corresponding of 3-
(4-benzoyl-1-aryl-1H-pyrazole-3-carbonyl)-1,5-diphenyl-1H-pyr-
azole-4-carbonitrile derivatives 32aec. The physical and spectral
data for obtained products are listed below.
4.1.10.4. 3-[3-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-1-(4-meth-
ylphenyl)-1H-pyrazole-4-carbonyl]-1,5-diphenyl-1H-pyrazole-4-car-
bonitrile (27c). Yield 73%; mp 281e282 ꢂC. IR (KBr) v cmꢀ1
:
2231, 2220 (2C^N), 1680, 1651 (2C]O). 1H NMR (DMSO-d6)
2.41 (s, 3H, CH3), 7.20e7.37 (m, 24H, ArH’s), 8.67 (s, 1H, pyr-
azole H-5); 13C NMR (DMSO-d6)
20.33 (Ar-4-CH3), 112.20,
d
d
116.73, 116.75 (pyrazole carbons), 119.73 (C^N), 120.53, 125.31,
125.94, 128.03, 128.48, 128.78, 128.86, 128.97, 130.53, 132.26,
137.35, 137.89, 148.23, 150.17 (pyrazole and aromatic carbons),
180.78, 182.31 (C]O). MS m/z (%): 700 (3.3), 456 (9.9), 428
(32.5), 272 (63.2), 244 (23.2), 77 (100). Anal. Calcd for
C44H28N8O2 (700.77): C, 75.42; H, 4.03; N, 15.99%. Found: C,
75.50; H, 4.07; N, 16.10%.
4.1.12.2. 3-(4-Benzoyl-1-phenyl-1H-pyrazole-3-carbonyl)-1,5-diphenyl-
1H-pyrazole-4-carbonitrile (32a). Yield 80%; mp 179e180 ꢂC. IR (KBr) v
cmꢀ1: 2237 (CN), 1680, 1650 (2C]O). 1H NMR (DMSO-d6)
d 7.23e7.50
(m, 20H, ArH’s), 8.89 (s, 1H, pyrazole H-5). MS m/z (%): 519 (Mþ, 13.0),
443 (49.7), 272 (20.7), 141 (13.2), 77 (100). Anal. Calcd for C33H21N5O2
(519.55): C, 76.29; H, 4.07; N, 13.48%. Found: C, 76.30; H, 4.10; N,
13.50%.
4.1.11. Reaction of 3-[3-(4-cyano-1,5-diphenyl-1H-pyrazole-3-yl)-
1-aryl-1H-pyrazole-4-carbonyl]-1,5-diphenyl-1H-pyrazole-4-
carbonitriles 27aec with hydrazine hydrate
4.1.12.3. 3-[4-Benzoyl-1-(4-chlorophenyl)-1H-pyrazole-3-carbonyl]-
1,5-diphenyl-1H-pyrazole-4-carbonitrile (32b). Yield 70%; mp
183e184 ꢂC. IR (KBr) v cmꢀ1: 2233 (CN), 1678, 1643 (2C]O). 1H
4.1.11.1. General procedure. Hydrazine hydrate (80%, 3 mL) was
added to a solution of the appropriate 3-[3-(4-cyano-1,5-diphenyl-
1H-pyrazole-3-yl)-1-aryl-1H-pyrazole-4-carbonyl]-1,5-diphenyl-
1H-pyrazole-4-carbonitrile 27 (5 mmol) in ethanol (10 mL). The
reaction mixture was heated under reflux for 1 h, concentrated in
vacuum, cooled, and diluted with water, the precipitate obtained
was filtered off, washed with ice-cold water, dried and crystallized
from ethanol. The synthesized compounds 29aec together with
their physical and spectral data are listed below.
NMR (DMSO-d6)
d
7.32e7.87 (m,19H, ArH’s), 9.11 (s,1H, pyrazole H-
112.35 (pyrazole), 119.68 (C^N), 124.86,
5); 13C NMR (DMSO-d6)
d
125.28, 125.34, 125.84, 127.08, 128.68, 128.78, 129.19, 129.29,
129.44, 129.53, 13.67, 133.13, 136.25, 137.44, 137.77, 137.81, 149.20,
150.01, 150.42, 154.08 (pyrazole and aromatic carbons), 180.46,
188.16 (C]O carbons). MS m/z (%): 555 (Mþ, 31.5), 553 (Mþ, 100),
180 (31.9), 141 (14.8), 77 (86.9). Anal. Calcd for C33H20N5O2Cl
(553.01): C, 71.54; H, 3.63; N, 12.64%. Found: C, 71.55; H, 3.65; N,
12.60%.
4.1.11.2. 3-[7-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-2-phenyl-
2H-pyrazolo[3,4-d]pyridazin-4-yl]-1,5-diphenyl-1H-pyrazole-4-car-
bonitrile (29a). Yield 68%; mp >300 ꢂC. IR (KBr) v cmꢀ1: 2225, 2235
4.1.12.4. 3-[4-Benzoyl-1-(4-methylphenyl)-1H-pyrazole-3-carbonyl]-
1,5-diphenyl-1H-pyrazole-4-carbonitrile (32c). Yield 73%; mp
142e143 ꢂC. IR (KBr) v cmꢀ1: 2233 (CN), 1678, 1650 (2C]O). 1H
(2C^N). 1H NMR (DMSO-d6)
d
7.20e7.47 (m, 25H, ArH’s), 8.60 (s,
1H, pyrazole H-5); 13C NMR (DMSO-d6)
d
111.87, 115.87 (pyrazole
carbons), 118.88 (C^N), 120.61, 120.6, 122.67, 125.41, 125.74, 127.04,
129.28, 129.50, 131.76, 133.38, 140.7, 150.09 (pyrazole and aromatic
carbons),152.68,159.65 (pyridazine C]N carbons). MS m/z (%): 682
(Mþ, 63), 180 (29), 141 (22.1), 77 (100). Anal. Calcd for C43H26N10
(682.74): C, 75.64; H, 3.83; N, 20.51; %. Found: C, 75.63; H, 3.81; N,
20.51%.
NMR (DMSO-d6)
d
2.45 (s, 3H, CH3), 7.22e7.60 (m, 19H, ArH’s), 9.02
26.24 (Ar-4-CH3), 112.23
(s, 1H, py H-5); 13C NMR (DMSO-d6)
d
(pyrazole), 119.46 (C^N), 124.46, 124.93, 125.23, 125.55, 125.75,
127.95, 128.67, 128.86, 129.20, 129.36, 129.52, 130.39, 132.40, 133.11,
137.26, 137.38, 137.58, 149.57, 150.01, 150.18, 153.98 (pyrazole and
aromatic carbons), 180.31, 187.92 (C]O carbons). Analysis for:
C34H23N5O2 (533.58): C, 76.53; H, 4.34; N, 13.13%. Found: C, 76.55;
H, 4.31; N, 13.15%.
4.1.11.3. 3-[7-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-2-(4-chlor-
ophenyl)-2H-pyrazolo[3,4-d]pyridazin-4-yl]-1,5-diphenyl-1H-pyr-
azole-4-carbonitrile (29b). Yield 72%; mp >300 ꢂC. IR (KBr) v
4.1.13. Reaction of 3-(4-benzoly-1-aryl-1H-pyrazole-3-carbonyl)-
1,5-diphenyl-1H-pyrazole-4-carbonitriles 32aec with hydrazine
hydrate
cmꢀ1: 2230, 2220 (2C^N). 1H NMR (DMSO-d6)
d
7.26e7.52 (m,
24H, ArH’s), 8.71 (s, 1H, pyrazole H-5); 13C NMR (DMSO-d6)
113.80, 114.21 (pyrazole carbons), 119.90 (C^N), 123.73, 125.51,
d
4.1.13.1. General procedure. Hydrazine hydrate (80%, 4 mL) was
125.75 126.13, 127.83, 129.20, 128.91, 129.29, 129.46, 129.68,
130.38, 135.05, 135.71, 138.22, 144.89 (pyrazole and aromatic
carbons), 156.13, 157.58 (pyridazine C]N carbons). MS m/z (%):
718 (Mþ, 25.2), 716 (Mþ, 77), 180 (33), 77 (100). Anal. Calcd for
C43H25N10Cl (717.19): C, 72.01; H, 3.51; N, 19.53%. Found: C,
72.10; H, 3.60; N, 19.50%.
added to a solution of appropriate 3-(4-benzoly-1-aryl-1H-
pyrazole-3-carbonyl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile 32
(5 mmol) in ethanol (10 mL). The reaction mixture was heated
under reflux for 1 h, concentrated in vacuum, cooled, and diluted
with water. The precipitate obtained was filtered off, washed with
water, dried and crystallized from ethanol. The synthesized

A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
5897
compounds 34aec together with their physical and spectral data
are listed below.
256, 409, 655 and 1050 mg/km B.W. After 24 h results were
recorded. The controls received tap water by gavage in the same
volume.
4.1.13.2. 3(2,4-Diphenyl-2H-pyrazolo[3,4-d]pyridazin-7-yl)-1,5-
diphenyl-1H-pyrazole-4-carbonitrile
(34a). Yield
68%;
mp
4.2.2.3. Observation. All animals were monitored continuously for
10 h after dosing for signs of toxicity. For the remainder of the 14
days study period, animals were monitored for mortality. At the
end of the study the number of dead animals was expressed in
percentage and the LD50 value was calculated according to Weill
(1952) method [42].
289e291 ꢂC. IR (KBr) v cm-1: 2320 (C^N). 1H NMR (DMSO-d6)
d
7.20e7.54 (m, 20H, ArH’s), 8.71 (s, 1H, pyrazole H-5). MS m/z: 515
(Mþ, 9.8), 244 (20.3), 77 (100). Anal. Calcd for C33H21N7 (515.57):
C, 76.87; H, 4.10; N, 19.01%. Found: C, 76.90; H, 4.13; N, 19.03%.
4.1.13.3. 3(2-(4-Chlorophenyl)-4-phenyl-2H-pyrazolo[3,4-d]pyridazin-
7-yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile (34b). Yield 79%; mp
4.2.2.4. Statistical analysis. The data were evaluated for homo-
geneity of variances and normality by Bartlett’s test. Bartlett’s
test indicated homogeneous variances, treated and control
groups were compared using a one-way analysis of variance
(ANOVA), followed by comparison of the treated groups to the
control groups by Dunnett’s t-test for multiple comparisons [43],
where variances were considered significantly different by Bar-
tlett’s test.
>300 ꢂC. IR (KBr) v cmꢀ1: 2240 (CN). 1H NMR (DMSO-d6)
d 7.45e7.88
(m, 19H, ArH’s), 9.34 (s, 1H, pyrazole H-5). MS m/z: 551 (Mþ, 13.1),
549 (Mþ, 35.1), 244 (13.0), 77 (100). Anal. Calcd for C33H20N7Cl
(550.01): C, 72.06; H, 3.67; N, 17.83%. Found: C, 72.02; H, 3.70; N,
17.80%.
4.1.13.4. 3(2-(4-methylphenyl)-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-
7-yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile (34c). Yield 74%; mp
>300 ꢂC. IR (KBr) v cmꢀ1: 2228 (C^N). 1H NMR (DMSO-d6)
d
2.42 (s,
4.2.3. In vitro antitumor screening
3H, CH3), 7.18e7.52 (m, 19H, ArH’s), 8.78 (s, 1H, pyrazole H-5). MS m/z:
529 (Mþ). Anal. Calcd for C34H23N7 (529.60): C, 77.11; H, 4.37; N,
18.51%. Found: C, 77.15; H, 4.35; N, 18.54%.
20 Compounds were subjected to the National Cancer Insti-
tute in vitro disease-oriented primary antitumor screening. 14
Cell lines of breast and ovarian tumor cell lines were utilized. The
human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum
4.2. Pharmacology
and 2 mM L-glutamine. For a typical screening experiment, cells
4.2.1. Antagonism of uterus weight increase due to estrogen
treatment (anti-estrogenic activity)
were inoculated into 96-well microtiter plates in 100 mL at
plating densities ranging from 5000 to 40,000 cells/well
depending on the doubling time of individual cell lines. After cell
inoculation, the microtiter plates were incubated at 37 ꢂC, 5%
CO2, 95% air, and 100% relative humidity for 24 h prior to addi-
tion of experimental drugs. After 24 h, two plates of each cell line
were fixed in situ with TCA, to represent a measurement of the
cell population for each cell line at the time of drug addition.
Experimental drugs were solubilized in DMSO at 400-fold the
desired final maximum test concentration and stored frozen prior
to use. At the time of drug addition, an aliquot of frozen
concentrate was thawed and diluted to twice the desired final
maximum test concentration with complete medium containing
50 mg mLꢀ1 gentamicin. Additional four 10-fold or 1/2 log serial
dilutions were made to provide a total of five drug concentra-
tions plus control. Aliquots of 100 mL of these different drug
dilutions were added to the appropriate microtiter wells already
containing 100 mL of medium, resulting in the required final
drug concentrations. Following drug addition, the plates were
incubated for an additional 48 h at 37 ꢂC, 5% CO2, 95% air, and
100% relative humidity. For adherent cells, the assay was termi-
nated by the addition of cold TCA. Cells were fixed in situ by the
gentle addition of 50 mL of cold 50% (w/v) TCA (final concen-
tration, 10% TCA) and incubated for 60 min at 4 ꢂC. The super-
natant was discarded, and the plates were washed five times
with tap water and air dried. Sulforhodamine B (SRB) solution
(100 mL) at 0.4% (w/v) in 1% acetic acid was added to each well,
and plates were incubated for 10 min at room temperature. After
staining, unbound dye was removed by washing five times with
1% acetic acid and the plates were air dried. Bound stain was
subsequently solubilized with 10 mM trizma base, and the
absorbance was read on an automated plate reader at a wave-
length of 515 nm. For suspension cells, the methodology was the
same except that the assay was terminated by fixing settled cells
at the bottom of the wells by gently adding 50 mL of 80% TCA
(final concentration, 16% TCA). The parameter used here is GI50
which is the log10 concentration at which PG is þ50, was
calculated for each cell line [44e46].
4.2.1.1. Animals. Immature female Sprague-Dawley rats weighing
about 55 g were obtained from Animal House Laboratory, Nile
Company, Cairo, Egypt and were acclimatized for 1 week in the
animal facility that has 12 h light/dark cycles with the temperature
controlled at 21e23 ꢂC. Normal rat chow and water were made
available.
The animals were housed individually in stainless steel cages in
temperature-controlled and humidity-monitored quarters. Test
animals were provided continuous access to tap water.
4.2.1.2. Procedure. Groups of 12 animals were injected daily for 7
days with estradiol 0.03e0.05
mg per animal S.C. and various doses
0.0e0.06 g per animal S.C. of the tested compounds and letrozole
m
as reference standard or estradiol alone.
The tested compound was administered in 0.5% solution of
carboxymethylcellulose (CMC) orally. On the 8th day, the animals
were sacrificed and the uterine weights were determined.
4.2.1.3. Evaluation. Mean values of each group were calculated and
expressed as % reduction of uterine weight compared to controls
treated with estradiol alone.
4.2.2. Evaluation of acute toxicity following single dose
administration
4.2.2.1. Animals. 600 Adult mice of both sexes weighing 25 ꢁ 3 g
were obtained from Animal House Laboratory, Nile company, Cairo,
Egypt and acclimatized for 1 week in the animal facility that has
12 h light/dark cycles with the temperature controlled at 21e23 ꢂC.
Normal rat chow and water were made available.
4.2.2.2. Procedure. LD50 was measured on 30 mice. Animals were
fasted for 12 h prior to dosing. Rats were divided into 6 groups,
5 animals in each group. Treatment rats were dosed by oral
gavage, using a curved, balltipped stainless steel feeding needle,
with aqueous suspensions of very fine powder of the tested
compound. Animals, in each group, were given doses of 100, 160,

5898
A.M. Farag et al. / European Journal of Medicinal Chemistry 45 (2010) 5887e5898
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Acknowledgment
[18] M.R. Shaaban, T.M.A. Eldebss, A.F. Darweesh,A.M.Farag, J.Chem. Res.(2010)8e11.
[19] A.M. Farag, A.S. Mayhoub, T.M.A. Eldebss, A.E. Amr, K.A.K. Ali, N.A. Abdel-
Hafez, M.M. Abdulla, Arch. Pharm. 343 (2010) 384e396.
[20] M.R. Shaaban, T.S. Saleh, A.M. Farag, Heterocycles 78 (2009) 151e159.
[21] M.R. Shaaban, T.S. Saleh, A.M. Farag, Heterocycles 78 (2009) 699e706.
[22] N.A. Kheder, Y.N. Mabkhot, A.M. Farag, Heterocycles 78 (2009) 937e946.
[23] A.M. Farag, N.A. Kheder, Y.N. Mabkhot, Heterocycles 78 (2009) 1787e1798.
[24] M.R. Shaaban, T.S. Saleh, A.S. Mayhoub, A. Mansour, A.M. Farag, Bioorg. Med.
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The authors are very grateful to the staff members of the
Department of Health and Human Services, National Cancer Insti-
tute (NCI), Bethesda, Maryland, USA, for carrying out the anticancer
screening of the newly synthesized compounds.
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